This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Metal-dependent histone deacetylases (HDACs) require Zn2+ or Fe2+ to regulate the acetylation of lysine residues in histones and other proteins in eukaryotic cells. The HDAC8 isozyme is perhaps the archetypical member of the class I HDAC family and represents the paradigm for understanding structure-activity relationships in this enzyme family. Here, we report the structures of HDAC8 complexes with trichostatin A and 3-(1-methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide (APHA) in a new crystal form. The structure of the APHA complex reveals that the hydroxamate C=O group does not coordinate to Zn2+ with favorable geometry, perhaps due to the constraints of its extended ? system, but instead accepts a hydrogen bond from Y306. Additionally, since APHA binds to only 2 of the 3 protein molecules in the asymmetric unit of this complex, the structure of the third monomer represents the first structure of HDAC8 in the unliganded state. Comparison of unliganded and liganded structures illustrates ligand-induced conformational changes in the L2 loop that likely accompany substrate binding and catalysis. Furthermore, these structures, along with those of the D101N, D101E, D101A, and D101L variants, support the proposal that D101 is critical for the function of the L2 loop. However, amino acid substitutions for D101 can also trigger conformational changes of Y111 and W141 that perturb the substrate binding site. We are continuing to work on getting higher resolution data for the D101E mutant, and working to explore potassium binding sites with the HDAC8 enzyme.